The long term objective of this project is the understanding of voltage-dependent gating of ion channels at the molecular level. In this proposal experiments are designed to describe structural aspects of Shaker and squid potassium channels and human skeletal muscle sodium channels. Cloned, engineered channels will be expressed in Xenopus oocytes and the function will be assessed with electrophysiological techniques while the structure will be probed with optical and chemical modification techniques. There are four specific aims. 1) Correlation of structural changes with the function of the voltage sensor. This will be approached using the technique of histidine scanning mutagenesis on the charges of the S4 and S2 segments. This technique utilizes protons to probe the accessibility of engineered histidine residues, usually replacing basic residues of the protein. In addition, fluorescent probes attached to specific sites of the channels (mutated to cysteine) will be used to assess changes in environment and correlate them with gating currents. 2) Measurements of distances in the channel molecule. This aim will use the fluorescence resonance energy transfer and its variant, lanthanide-based resonance energy transfer, to measure distances between specific sites across subunits or between an specific site in the channel and an specific toxin sitting on the pore of the channel. The sites of attachment of the fluorophores and lanthanides are engineered cysteines in the channel molecule and Agitoxin II. Distance measurements will be done on sites in the S2, S3 and S4 segments using a newly developed optical setup that allows simultaneous voltage clamp and accurate measurements of gating currents. Distance measurements will be performed at different membrane potentials to assess possible distance during activation of the conductance. 3) Study of the activation and inactivation pathways. In this aim a study of the initial fast event of gating and a detailed characterization of the events leading to channel opening and slow inactivation will be studied with noise analysis of gating currents in the Shaker K channel and with gating currents in the Sodium channel to correlate them with the structural information obtained in aims 1 and 2. 4) Modeling. Kinetic modeling will be done to account for the results in electrophysiologyical and optical experiments. Simulations of fluctuations produced by voltage ramps will be compared to the noise analysis experiments of aim 3 to test models of activation and inactivation. In addition, molecular modeling will be done based on the results of distance measurements, including possible distance changes occurring during activation. These experiments are expected to give us insight on the molecular rearrangements concomitant with voltage-dependent gating, which is a basic property of many membrane channels and it has critical importance in excitability and cell homeostasis.